Insights into the mechanism of SARS-CoV-2 main protease autocatalytic maturation from model precursors

A critical step for SARS-CoV-2 assembly and maturation involves the autoactivation of the main protease (MProWT) from precursor polyproteins. Upon expression, a model precursor of MProWT mediates its own release at its termini rapidly to yield a mature dimer. A construct with an E290A mutation within MPro exhibits time dependent autoprocessing of the accumulated precursor at the N-terminal nsp4/nsp5 site followed by the C-terminal nsp5/nsp6 cleavage. In contrast, a precursor containing E290A and R298A mutations (MProM) displays cleavage only at the nsp4/nsp5 site to yield an intermediate monomeric product, which is cleaved at the nsp5/nsp6 site only by MProWT. MProM and the catalytic domain (MPro1-199) fused to the truncated nsp4 region also show time-dependent conversion in vitro to produce MProM and MPro1-199, respectively. The reactions follow first-order kinetics indicating that the nsp4/nsp5 cleavage occurs via an intramolecular mechanism. These results support a mechanism involving an N-terminal intramolecular cleavage leading to an increase in the dimer population and followed by an intermolecular cleavage at the C-terminus. Thus, targeting the predominantly monomeric MPro precursor for inhibition may lead to the identification of potent drugs for treatment.


Figure S1 .
Figure S1.Amino acid sequence of recombinant MPro constructs used in this study and their designations.Non-native residues flanking the sequences are underlined.Mutation sites are shown in red.Theoretical mass of the purified protein is indicated below the sequence of the corresponding construct.(-25)and(-102)  denote 25 and 102 amino acids of the C-terminal residues of nsp4, flanking the N-terminus of nsp5 (MPro), appended to MPro. (+3) -GB1 denotes 3 Nterminal residues of the nsp6 sequence (SAV) followed by 56 residues of GB1 and 6His-tag.The 6H-tag at the C-terminus of Precursor WT and its analogues enables isolation by Nickel-Affinity Chromatography (NAC) and characterization of the intermediate precursor (MPro M-IP ) containing the C-terminal flanking sequence, which results from N-terminal cleavage at the nsp4/nsp5 site.As the size difference between the two is small (4.9 kDa), excluding the 6H-tag in construct(-

Figure S2 .
Figure S2.N-terminal autoprocessing of MPro precursor mimetics in E. coli.These panels are reproduced from our previous publication 3 solely for ease of comparison with the results presented in this work.The N-terminal cleavage site is indicated with a downward black arrow.The gels show the time course of the autoprocessing reaction of MPro WT (a), MPro M (b) and MPro 1-199 (c) precursor constructs with flanking nsp4 sequences as indicated.Cells (12 ml) were harvested at the indicated time points, and equal volumes of the bound fractions following NAC were analyzed by SDS-PAGE.The precursor, products released upon cleavage at the N-terminus of MPro and molecular weight standards (M) are indicated in kDa.

Figure S3 .
Figure S3.Estimation of MPro M-IP dimer dissociation constant by SV-AUC.(a) Normalized sedimentation velocity absorbance c(s) distributions for various concentrations of MPro M-IP in the presence of 2-fold molar excess GC373 support an inhibitor induced monomer-dimer selfassociation.M and M/D denote monomer and monomer/dimer equilibrium boundary, respectively.(b-e)Sedimentation data collected at 50,000 rpm and 25 °C over 7 hours at 3.6 to 32 µM MPro M- IP were analyzed globally in terms of a reversible monomer-dimer self-association using Lamm equation modeling (see legend to Fig.S5).The analysis returns a Kdimer of 8.5 ± 1.5 µM.

Figure S5 .
Figure S5.Estimation of the dimer dissociation constant of MPro R298A and MPro E290A by SV-AUC.Sedimentation velocity absorbance c(s) distributions at the indicated loading concentrations for (a) MPro R298A , (b) MPro E290A and (c) MPro E290A in the presence of GC373.(d) Sedimentation velocity data collected at 50,000 rpm and 25°C in 3 mm pathlength cells with scans collected over 6 hours.The data were analyzed globally in terms of a reversible monomer-dimer self-association model using Lamm equation modeling.For clarity only every sixth scan and every third experimental data point are shown.Best-fits are represented by a solid line through the experimental points.A bitmap representation of the residuals to the best-fit, together with the combined residuals, are shown below each plot.The analysis returns a monomer-dimer dissociation constant (Kd) of 4.8 ± 0.6 µM for MPro E290A with GC373.Based on the loading concentration and dissociation constant, the dimer contribution (in monomer units) was determined to be 48% at 4.3 µM MPro E290A in the presence of 2x GC373.M and M/D denote monomer and monomer/dimer equilibrium boundary, respectively.

Figure S6 .
Figure S6.Comparison of MPro WT and MPro M structures.(a) Superposition of MPro M and MPro WT complexes with GC373 near E290A mutation site demonstrating the loss of the E290…R4' salt bridge.In MPro WT -GC373 Glu290 has two alternate conformations with occupancies 46 and 54 %.(b) Superposition of MPro M -GC373 (colored deep olive, this work, PDB ID 8FIG), MPro WT -GC373 (colored salmon, PDB ID 7UKK 3 ) and inhibitor-free MPro WT (colored gray, PDB ID 7JUN 4 ) cartoon representations near the R298A mutation site shows a conformational reorientation of the C-terminal residues (black curved arrow).Residues beyond the site of mutation at position 298 are colored cyan for MPro M and dark gray for MPro WT .